The overall objective of this grant application is to understand the mechanism of RNA synthesis of mouse hepatitis virus (MHV), a murine coronavirus. During the last grant period, our laboratory has established two unique mechanisms associated with MHV RNA synthesis, leader-primed transcription of subgenomic mRNAs and high-frequency RNA recombination. These phenomena suggest that MHV RNA synthesis is discontinuous and nonprocessive. We have also started to characterized the structure and properties of the virus-specific RNA polymerases, and have found an autoproteolytic activity associated with the precursor of the RNA polymerase. These data, along with an unusually large size of the possible RNA polymerase gene (more than 23 kb) and the large size of MHV RNA genome (32 kb), indicate that MHV RNA synthesis is extremely complex. This grant application proposes to continue our studies on the mechanism of MHV RNA synthesis. Four projects will be carried out: (1) Identification of natural transcriptional initiation sites on MHV genome. We will characterize the transcriptional initiation sequences of the minor mRNAs and "anomalous" mRNA species which are expressed only in some MHV strains. Both leader RNAs and template sequences at the transcriptional initiation sites will be studies; (2) Development and characterization of an in vitro transcription system for MHV. We will use a lysolecithin permeabilization method to establish an in vitro system to study the detailed mechanism of leader-primed transcription and the sequence requirement for both the leader RNA and transcriptional initiation sites; (3) Characterization of the polyprotein encoded by gene 1 of MHV. This protein probably is the precursor to the virus-specific RNA polymerases. We will study its gene sequence and protein processing. We will also attempt to identify the enzymatic activities, including proteases and helicase activities associated with these proteins. Finally, we will study whether these proteins can complement temperature-sensitive mutants which are defective in RNA synthesis; and (4) Characterization of transcriptional and post-transcriptional regulation of gene 2b expression. We will particularly study the mechanism by which A59 strain synthesizes gp65 despite the fact that the virus does not have a complete open reading frame for this protein. These projects are expected to shed further light on the mechanism of RNA synthesis and gene regulation in MHV.